The study was to determine the long-term effects of subtropical monoculture and rotational cropping systems and fertilization on soil enzyme activities and soil C, N, and P levels. Cropping systems included continuous sorghum(Sorghum bicolor L.), cotton(Gossypium hirsutum L.), corn(Zea mays L.), and cotton/sorghum rotations after 26 years of treatment imposition. Soil under continuous sorghum and continuous corn had 15% and 11%, respectively, greater C concentrations than soil under continuous cotton.Organic C was 10% higher at 0–7.5 cm than at 7.5–15 cm. Total N followed similar trends with soil depth as organic C. Continuous sorghum had 19% higher total N than other crop species and rotations. With fertilization, continuous cotton had the highest total P at 0–7.5 cm and sorghum had the highest at 7.5–15 cm. Soil total P was 14% higher at 0–7.5 than at 7.5–15 cm, and fertilization increased 15% total P compared to unfertilized soil. Arylsulfatase, alkaline phosphatase, and β-d-glucosidase activity were the highest for sorghum and the lowest for cotton. Rotation increased enzyme activities compared to continuous cotton but not for continuous sorghum. Of all crop species and rotations, continuous cotton generally showed the lowest levels of organic matter and enzyme activities after 26 years. Fertilization significantly increased the yields for all cropping systems, but rotation had no significant effect on either sorghum or cotton lint yield compared to each crop grown in monoculture. Long-term cropping did not increase soil organic matter levels beyond short-term gains, indicating the difficulty in promoting C sequestration in subtropical soils. The study was to determine the long-term effects of subtropical monoculture and rotational cropping systems and fertilization on soil enzyme activities and soil C, N, and P levels. Cropping systems included continuous sorghum (Sorghum bicolor L.), cotton (Gossypium hirsutum L .), corn (Zea mays L.), and cotton / sorghum rotations after 26 years of treatment imposition. Soil under continuous sorghum and continuous corn had 15% and 11%, respectively, greater C concentrations than soil under continuous cotton. Organic C was 10% higher at 0-7.5 cm than at 7.5-15 cm. Total N followed similar trends with soil depth as organic C. Continuous sorghum had 19% higher total N than other crop species and rotations. With fertilization, continuous cotton had the Soil total P was 14% higher at 0-7.5 than at 7.5-15 cm, and fertilization increased 15% total P compared to unfertilized soil. Arylsulfatase , alkaline phosphatase, and β-d-glucosidase activity were the highest for sorghum and the lowest for cotton. Rotation increased enzyme activities compared to continuous cotton but not for continuous sorghum. Of all crop species and rotations, continuous cotton generally showed the lowest levels of organic matter and enzyme activities after 26 years. Fertilization significantly increased the yield for all cropping systems, but rotation had no significant effect on either sorghum or cotton lint yield compared to each crop grown in monoculture. Long-term cropping did not increase soil organic matter levels beyond short- term gains, indicating the difficulty in promoting C sequestration in subtropical soils.